Alleviation of the memory deficits and memory components of psychiatric dysfunctions by altering atypical PKM activity

ABSTRACT

Methods have been developed for alleviating memory problems or psychiatric dysfunctions that have a memory formation component. These methods are based on the finding that a truncated form of an aPKCζ protein is intimately involved in memory formation in animals. This finding is also central to methods for determining drugs that will have an effect on memory formation or the memory formation component of psychiatric dysfunctions.

RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/326,948, filed on Oct. 4, 2001, and also claims thebenefit of U.S. Provisional Application No. 60/287,165, filed on Apr.27, 2001.

[0002] The entire teachings of the above application(s) are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

[0003] Mental illnesses are significant for their debilitating effect onthe individuals who suffer their manifestations. The rest of society issignificantly affected since they must provide care for the individualswho are afflicted with these diseases. Mental impairments that resultfrom such events as strokes are also significant for their adverseconsequences for the individuals who suffer their debilitating effects.Memory deficits in individuals have major consequences for them and forothers with whom they interact. Although much effort has been expendedin an attempt to overcome these maladies, there remain largedeficiencies in the ability to understand and effectively treat them.Further treatment modalities are constantly being sought. In particular,the biochemical bases for these diseases or states remains largelyunknown and knowledge of these bases will foster further drugdiscoveries.

[0004] All of these diseases or states have a significant memorycomponent. This is particularly true, obviously, when memory loss orabnormally poor memory is the prime or only symptom. Knowing biochemicalmolecules that centrally participate in memory will lead to methods andcompositions that lessen memory abnormalities and will alleviate mentaldiseases or psychiatric dysfunctions by addressing the memory componentof these diseases or dysfunctions.

SUMMARY OF THE INVENTION

[0005] The present invention is directed to methods of alleviatingmemory problems and psychiatric dysfunctions in mammals by modulatingthe expression, or activity, of a truncated form of an aPKC ZETA (aPKCζ)protein in the central nervous system of the mammal. A particulartrucated form of the aPKCζ protein that is responsive to modulation isaPKMζ. Modulation of protein expression can be achieved by eitherinduction or inhibition of formation of aPKMζ. Modulation of activitycan be achieved by affecting interacting proteins that participate inthe normal memory formation process. Memory problems include abnormalmemory formation due to normal aging, injury to the brain,neurodegeneration, and Alzheimer's disease or other decreases incognitive ability. The present invention alleviates memory deficitsassociated with these situations. In this instance, the problem isalleviated by inducing expression of aPKMζ and memory formation isparticularly improved when the expression of aPKMζ is induced.Psychiatric dysfunctions for which alleviation can be achieved includeattention deficit disorder, autism, fragile X syndrome, bipolardisorder, schizophrenia, obsessive compulsive disorders and phobias. Inthis regard, the psychiatric dysfunctions are alleviated by affecting oraltering the memory formation component of the particular dysfunction.

[0006] The present invention is also directed to methods of identifyingsubstances that can affect memory formation or psychiatric dysfunctionsin mammals. In these methods, a substance under scrutiny is administeredto a mammal. It is then determined whether the substance alters theexpression or activity of aPKMζ protein in the central nervous system ofthe mammal when compared to the expression or activity of the sameprotein in the absence of the substance. If there is a difference insuch expression or activity, and the aPKMζ protein is associated with amemory defect or a given psychiatric dysfunction, the substance affectsthat defect or dysfunction. The portion of the psychiatric dysfunctionthat is affected when the substance alters the expression or activity ofthe aPKMζ protein is the memory formation component of the dysfunction.In particular instances, an increase in expression or activity of theaPKMζ protein when the substance is administered is indicative that thesubstance will enhance the memory formation component of dysfunction. Bycontrast, a decrease in expression or activity of the aPKMζ protein whenthe substance is administered is indicative that the substance willinterfere with the memory formation component of the dysfunction.

[0007] The present invention is further directed to methods forassessing the effect of a drug on regular memory disorders or the memoryformation component of a psychiatric dysfunction. To test the effect ofa drug on regular memory disorders, animal models for a memory disorderare trained and tested in the presence and absence of the drug. If thedrug affects performance relative to the performance of animals thatwere trained identically but in a drug-free state, then the drug has aneffect on regular memory disorders. Regular memory disorders result fromthe processes of normal aging, traumatic injury to the brain,Alzheimer's disease, and neurodegeneration. To test the effect of thedrug on the memory formation component of a psychiatric dysfunction, thedrug is administered to an animal that has an animal model for aspecified psychiatric dysfunction. The animal is subjected to a trainingprotocol and the performance index of the animal is assessed. The drugis found to have an effect on memory formation when the performancesignificantly differs between drug-free and drug-treated animals thatwere trained identically. Psychiatric dysfunctions which have a memoryformation component and for which the effects of drugs can be assessedby these methods include attention deficit disorder, autism, Fragile Xsyndrome, bipolar disorder, schizophrenia, obsessive compulsive disorderand phobias.

[0008] The present invention is further directed to methods ofalleviating regular memory disorders and psychiatric dysfunctions inmammals by modulating the expression of an aPKMζ gene that is associatedwith the regular memory disorder or the psychiatric dysfunction. Whenthe modulation is induction of the aPKMζ gene, the induction enhancesthe normal memory formation process, or the memory formation componentof a psychiatric dysfunction. Regular memory disorders result fromnormal aging, traumatic injury to the brain, Alzheimer's disease andneurodegeneration. Psychiatric dysfunctions for which alleviation can beachieved by modulation of an aPKMζ gene include attention deficitdisorder, autism, Fragile X syndrome, bipolar disorder, schizophrenia,obsessive compulsive disorder, and phobias.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1A Memory enhancement by MaPKMζ in Drosophila. Memoryenhancement by heat-shock induction after training of two independentlines bearing a hsp 70-MaPKMζtransgene. Flies were subjected tosingle-cycle training, allowed to recover at 25° C., and the MaPKMζtransgene was induced with a 30-min, 32° C. heat shock. Performance wasmeasured 24 h later. The induction of line 14 and the wild-type (WT)control began 1 h after training ended, and the induction of line 43began 30 min after training. Both lines show clear induction effects(n=8 for all groups). Error bars represent standard error of the meanand, unless noted, the asterisks indicate statistical significancecalculated using ANOVA and Dunnet's test throughout this work.

[0010]FIG. 1B. Temporal specificity of the memory enhancement by MaPKMζ.MaPKMζ transgenic flies (line 14) were subjected to a 30-min, 25-32° C.heat shock at various times before and after a single cycle of training,and tested for 24-h memory. Histograms from left to right: heat shockended 1 h before, 30 min after, 1 h after or 2 h after training began;heat shock began 30 min, 1 h after or 2 h after training ended,respectively, n=8 for all groups.

[0011]FIG. 2A. Memory enhancement requires persistent kinase activityand is not due to sensory enhancement. Neither a kinase-inactive (KI)mutant of MaPKMζ nor full-length (FL) MaPKMζ enhances 24-h memory. Twoindependent lines (FL-3A, FL-14A) of the full-lengthhsp70-MaPKMζtransgene (Methods) and two independent lines (KI-1 andKI-2) of a hsp70-KI-MaPKMζtransgene were assessed for memoryenhancement. The KI-MaPKMζ mutant (K281W) disrupts kinase activity byaltering the ATP-binding domain. Two different heat-shock schedules wereused: a 30-min, 32° C. induction 3 h before training, or a 30-min, 32°C. heat shock given 30 min after the end of training. Neither heat-shockregimen produced induction-dependent enhancement of 24-h memory in anyof these lines.

[0012]FIG. 2B. Memory enhancement requires persistent kinase activityand is not due to sensory enhancement. Induction of MaPKMζ does notaffect peripheral behaviors. Flies were subjected to a 30-min, 32° C.heat shock. allowed to recover for 1 h, then tested for shockreactivity. Olfactory acuity was tested by exposing the flies to a30-min, 32° C. heat shock, and assayed for odor acuity 24 h later. Thelegend inset in the olfactory acuity panel applies to both sets ofhistograms. In all measures, both the MaPKMζ lines (14 and 43) wereindistinguishable from the wild-type controls. The most critical controlis the 10⁻² olfactory acuity test, and for this the transgenic flieswere heat-shocked (or not), stored, and tested simultaneously with theirrespective wild-type controls. Both sets of wild-type controls, adjacentto their transgenic counterparts, were included for direct comparison.

[0013]FIG. 3A. Expression and biochemical analyses of transgenic lines.Western blot analyses of MaPKMζ and MaPKMζ induction after heat shock.Flies were subjected to heat shock (32° C. or 37° C.) for 30 min. Top,time-course western blots from both MaPKMζ lines. At the indicated timepoints, flies were collected and head extracts were used for westernblot analysis. Middle, direct comparison of the induction achieved at32° C. versus 37° C. In each case, the flies were heat-shocked for 30min, and head extracts were made after 3 h of recovery time. Bottom,direct comparison of the expression of the MaPKMζ (line 14), thekinase-inactive (KI)-MaPKMζ (KI-1), and full-length (FL)-MaPKMζ(FL-14A). For each western blot, equivalent amounts of total protein(five heads per lane) were loaded in each lane.

[0014]FIG. 3B. Expression and biochemical analyses of transgenic lines.Induction of MaPKMζ results in an increase in a typical PKC activity infly head extracts. MaPKMζ transgenic flies (line 43) were subjected to a37° C. heat shock for 30 min, allowed to recover at 25° C. for 1 h, andfrozen in liquid nitrogen. Control flies remained at 25° C. throughout.Total protein extracts were made from fly heads and assayed for PKCactivity. An increase in kinase activity could be detected (−Ca²⁺/−PMA:HS+versus −HS, p=0.0057, n=4; +Ca²⁺/+PMA: HS+versus —HS, p=0.0011, n=4:Student's t-test) with a strong (37° C.) but not with a mild (32° C.)heat shock (data not shown). The induction levels are much greater at37° C. versus 32° C. (a).

[0015]FIG. 4A. MaPKMζ induction enhances 4-day memory after massed, butnot spaced training. MaPKMζ induction after massed training enhances4-day memory. MaPKMζ flies were trained with 10 cycles of massedtraining, allowed to rest for 30 min, then subjected to a 30-min heatshock (32° C.). Performance was measured at 4 days; n=8 for all groups.Both lines (14 and 43) show significant induction-dependent improvementof 4-day memory. At 4 days, the memory induced by massed training hasnormally decayed so that the PI=0, as is the case for uninduced flies(-HS).

[0016]FIG. 4B. Four-day memory produced by spaced training is notimproved by MaPKMζ induction. MaPKMζ flies were trained with 10 cyclesof spaced training (Methods), allowed to rest for 30 min, and subjectedto a 30-min, 32° C. heat shock. Memory was measured 4 days aftertraining. n=8 for all groups. Neither line shows a significant effect oftransgene induction on memory after spaced training. To rule out aneffect of saturation, flies were also trained with a submaximal numberof spaced trials (7), but transgene induction still had no effect (datanot shown).

[0017]FIG. 5. MaPKMζ induction corrects the memory deficit of radishmutants. Homozygous radish females were mated to males homozygous for anautosomal MaPKMζ transgene. (Line 43 was used in this experiment.) Maleand female progeny from this mating were trained and tested together,providing an internal control, and then counted separately to generatesex-specific PI values. The genotypes of the males and females are givebelow the histogram. Using Dunnet's test with uninduced females as thecontrol group, the only group showing statistically differentperformance is the uninduced males (noted by the asterisk). Analysis ofHS+versus HS− groups using Student's t-test shows that both males andfemales display significant differences in performance by transgeneinduction (denoted by #).

[0018]FIG. 6A. A Drosophila homolog of MaPKMζ is present and active inDrosophila head extracts. Antisera against MaPKC/Mζ detect DAPKC andDaPKM. Separate extracts from fly heads and bodies were analyzed onwestern blots using antisera directed against the C-terminal 16 aminoacids of MaPKC/Mζ. The molecular weights of the two immunoreactive bandsagree with those predicted for the DaPKC (73 kDa) and DaPKM (55 kDa)isoforms. The putative DaPKM is enriched in heads.

[0019]FIG. 6B. DaPKC and DaPKM immunoreactivity is competed withDaPKC-specific peptides. Western blots were done on head extracts. Theantiserum was added along with 0, 1-, 10-, 100- or 1000-fold excessDaPKC-590 peptide. This peptide is derived from the C terminus of theDaPKC protein. The antiserum was made against the C terminus of MaPKMζ.which is homologous to the DaPKC protein. A peptide derived from anupstream region of DaPKC (545) does not compete with theimmunoreactivity even at 1,000-fold excess.

[0020]FIG. 6C. Atypical PKC activity is enriched in extracts made fromDrosophila heads. Separate extracts were made from wild-type fly headsand bodies as in (a). They were assayed for aPKC activity as in FIG. 2b.

[0021]FIG. 7A. Chelerythrine treatment or KI-MaPKMζ expression inhibits24-h memory produced by massed training, but not learning, inDrosophila. Chelerythrine inhibits 24-h memory after massed training ina dose-dependent manner. Wild-type flies were fed either sucrose (0 μM)or 25 μM, 50 μM or 100 μM chelerythrine in a sucrose solution. They weregiven three cycles of massed training and assessed for 24-h memory. n=8for all groups.

[0022]FIG. 7B. The KI-MaPKMζ mutant inhibits 24 h memory produced bymassed training. Each line of the hsp70-KI-MaPKMζtransgene (KI-1 andKI-2) was induced with a 30-min, 37° C. heat shock, allowed to recoverfor 3 h at 25° C., and then given 10 cycles of massed training. LineKI-1 induction caused a significant reduction in 24-h memory, andinduced KI-1 flies were also significantly different from inducedcontrol flies (WT, HS+). Line KI-2 induction also seems to reducememory, but this line may not be as effective as KI-1. n=8 for allgroups.

[0023]FIG. 7C. Neither chelerythrine nor KI-MaPKMζ induction affectslearning. After a 1-h starvation period, wild-type flies were fed eithersucrose (0 μM) or 100 μM chelerythrine in a sucrose solution for 3 h.The KI-MAPKMζ was induced as in (b). In both cases. the flies were thengiven single-cycle training and tested immediately after training toassess learning. There was no difference between relevant groups:sucrose-fed flies were not different from chelerythrine-fed flies, andWT HS+flies were not different from KI-1 HS+or KI-2 HS+flies. n=8 forall groups.

[0024]FIG. 8A DaPKM induction enhances memory. DAPKM enhances 24-hmemory after single-cycle training. Flies were given single-cycletraining, allowed to recover at 25° C. for 30 min, and the DaPKMtransgene was induced by a 32° C. heat shock lasting 30 min. Performancewas measured 24 h later. Three independent transgenic lines were tested(FI9, M40, and M78). n=8 for all groups. The asterisks indicatesignificant differences.

[0025]FIG. 8B. DaPKM enhances 4-day memory after massed training.DaPKM-M78 flies were trained with 10 cycles of massed training, allowedto recover for 30 min at 25° C., and induced as in (a). Performance wasmeasured at 4 days; n=8 for all groups. Only line M78 was used for thisanalysis, and it shows significant induction-dependent improvement of4-day memory.

[0026]FIG. 8C. Western blot analyses of DaPKM induction afterheat-shock. Flies were subjected to heat shock (32° C.) for 30 min.Left, time-course western blots of the DaPKM-M78 line. At the indicatedtime points, flies were collected and head extracts were used forwestern blot analysis. All times are relative to the end of theheat-shock treatment. Right, direct comparison of the induction of theM40 and M78 lines. In both panels, the upper band is a background bandincluded as a load-control reference.

DETAILED DESCRIPTION OF THE INVENTION

[0027] This invention relates to methods of alleviating memory problemsand psychiatric dysfunctions in mammals, particularly in humans, bymodulating the expression or activity of a truncated form of an aPKCζprotein in the central nervous system of the mammal. The expression ofthe truncated form of the aPKCζ protein is modulated by affectingmolecular processes of transcription, mRNA stability, protein stability,proteolytic processing, and translation initiation. The activity of theprotein is modulated by post-translational modifications on the protein,protein:protein interactions, and subcellular localization of theprotein.

[0028] Modulation of expression occurs when the amount of the truncatedform of the aPKCζ protein differs from the amount that is presentwithout modulation. There can be an increase in the amount of truncatedform of the aPKCζ protein, decrease in the amount of truncated form ofthe aPKCζ protein, or a variation over a defined time span of anincrease and/or a decrease in the amount of truncated form of the aPKCζprotein. For example, the amount of truncated form of the aPKCζ proteincan increase in a linear or nonlinear fashion over time, decrease in alinear or nonlinear fashion, or alternatively increase and decrease,linearly or nonlinearly. An increase in the amount of the truncated formof the aPKCζ protein, or in the amount of an inhibiting form of theprotein, in a linear or nonlinear fashion, is generally preferred forthe alleviation of psychiatric dysfunctions.

[0029] The protein that is formed by the expression process in thisinvention is a truncated form of an aPKCζ protein. This protein isproduced either by synthesis of the full-length protein, followed byproteolytic processing, or de novo synthesis of the truncated form ofthe protein, aPKMζ. This de novo synthesis may occur from transcriptioninitiation sites located in intronic regions of the gene, and/ortranslation initiation from internal methionine codons.

[0030] Modulation of the expression of the truncated form of an aPKCζprotein can occur either by an induction or by an inhibition of theexpression of the truncated form. When induction of expression occurs,more of the truncated form is produced than when this induction is notpresent. When inhibition of expression occurs, less of the truncatedform is produced than when this inhibition is not present. Under certainconditions, a given amount of the truncated form of the aPKCζ proteinnormally may be produced. Under these conditions, induction ofexpression increases the amount of the truncated form that is producedand inhibition of expression decreases the amount of the truncated formthat is produced from this given amount. In these instances, modulationof the expression of the truncated form causes an increase, decrease, oralternative increase and decrease, linearly or nonlinearly, from thegiven amount that is normally present.

[0031] In this invention, the preferred truncated form of the aPKCζprotein is aPKMζ. This is the truncated form of the a typical isozymePKCζ that lacks the N-terminal regulatory domain of the PKCζ protein.The N-terminal regulatory domain contains a pseudosubstrate region aswell as binding sites for the required cofactors. The aPKMζ, which lacksthis N-terminal regulatory domain, contains the C-terminal catalyticdomain and is a persistently active kinase derived from the aPKCζisozyme. Induction of the expression of aPKMζ protein or inhibition ofthe expression of the aPKMζ protein are preferred in this invention toalleviate psychiatric disorders. Of these, induction of expression ismost preferred.

[0032] Normal memory problems can be alleviated by modulating theexpression or activity of aPKMζ. These memory problems can result fromnormal aging, traumatic injury to the brain, Alzheimer's disease andneurodegeneration. Classical memory disorders include loss or lack ofability to recall specific past experiences or events. The loss or lackof ability to make proper or normal associations between these priorevents or past experiences is also included in these disorders. Thesedisorders also include the loss or lack of ability to make proper ornormal associations between prior events or past experiences and presentcognitive functions or experiences. Short term memory loss and long termmemory loss are particular memory deficits that are included in thesedisorders. Short term memory losses are the loss or lack of ability torecall or make correct or proper associations between presentperceptions and recent events or experiences. Long term memory lossesare the loss or lack of ability to recall or make correct or properassociations between present perceptions and events or experiences thatwere perceived some time ago by the individual. The distinction betweenshort term memory and long term memory varies with the animal species,behavioral task, and training regimen, and is generally known to peopleto whom this distinction is important. For all animals, long-term memoryis the memory phase whose induction is sensitive to protein synthesisinhibitors given acutely around the time of training. Short-term memoryare all phases of memory that are resistant to such inhibitors.Generally, short term memories last from minutes, to hours and a fewdays after training, while long-term memories persist for longer periodsof time.

[0033] Many memory problems and psychiatric dysfunctions can bealleviated by modulating the expression of aPKMζ in animals. Alleviationof a given psychiatric dysfunction occurs when the symptoms of thedysfunction are lessened and the individual exhibits more normalbehavior patterns and modes. In certain instances, and usually desired,alleviation of a given psychiatric dysfunction is essentially completeand the individual exhibits normal behavior. In rare instances, theindividual exhibits normal behavior traits before the expression ofaPKMζ protein is modulated. Under these circumstances, better thannormal behavior is sought and expression of the truncated form ismodulated to achieve this result.

[0034] Among the memory problems that can be alleviated by modulatingthe expression of aPKMζ are those resulting from normal aging, injury tothe brain, Alzheimer's disease or neurodegeneration. Among thepsychiatric dysfunctions that can be alleviated by modulating theexpression of aPKMζ are attention deficit disorder, autism, fragile Xsyndrome, bipolar disorder, schizophrenia, obsessive compulsivedisorder, and phobias.

[0035] In the present invention, memory is affected by modulation ofexpression of the aPKMζ protein. Depending upon the protein isoform thatis modulated, memories can be enhanced or blocked. The aPKMζ appears tohave a noticeable effect on short term memory when its expression ismodulated. Short term memory improves when expression of aPKMζ isinduced.

[0036] Without being bound by any mechanism of action, it appears thatthe psychiatric dysfunctions that are alleviated by modulating theexpression of a truncated form of aPKMζ in the central nervous system ofthe animal are aided or relieved because the modulation affects thememory formation component of the psychiatric dysfunction. By making theappropriate changes to the memory formation component, the symptoms ofthe psychiatric dysfunction are lessened and the psychiatric dysfunctionis altered in a favorable manner. In many instances, it is the shortterm memory component of the psychiatric dysfunctions that is affectedby modulating the expression of the APKMζ protein. Induction ofexpression of the aPKMζ causes an enhancement of the short term memorycomponent of the psychiatric dysfunction. Inhibition of expression oractivity of aPKMζ causes an interference with the short term memorycomponent of the psychiatric dysfunction. Either an enhancement orinterference with the short term memory component can alleviate a givenpsychiatric dysfunction. Whichever process is desired to alleviate thepsychiatric dysfunction will be employed. For example, when anindividual exhibits a lack or loss of short term memory ability, theexpression of aPKMC can be induced to relieve symptomology.

[0037] The amount of truncated form of aPKCζ protein in the centralnervous system of the animal can be changed or modulated in a variety ofmanners. Transcription of the endogenous gene can be modulated. Anysmall molecule or physiological stimulus that affects the amounts oractivity of the different transcription factors that modulate geneexpression will affect levels of the mRNA and protein. Alternatively,DNA based manipulations can be performed to change the regulation of theendogenous gene. Exogenous regulatory sequences can be added to theendogenous gene, putting the gene under the control of different DNAsequences, proteins that bind those sequences, and effectors that affectthose proteins. In these situations, modulation of expression of thetruncated form occurs when the effector is administered from an externalsource or withheld, depending on the action that occurs at theregulation site.

[0038] Another manner of changing or modulating the amount of aPKMζprotein in the central nervous system of the animal is using transgenictechnology. A transgene that encodes a desired aPKMζ protein is insertedinto the genome of the animal. The transgene can be inserted usingrecombinant techniques recognized and known to skilled persons such asmolecular biologists. The transgenic animal can contain one or morecopies of the transgene that encodes the truncated form of the aPKMζprotein. This transgene may contain the endogenous gene. More likely,the transgene encodes a selected aPKMζ protein of another animalspecies. In either instance, the transgene can be under the control ofeither endogenous regulation sites or regulation sites obtained fromexogenous sources. Endogenous regulation sites can be employed when thetransgene is inserted at an appropriate locus in the genome where geneexpression is controlled by the endogenous regulation site. However,regulation sites from exogenous sources are more often employed whentransgenes are used. The regulation sites are often easier to includewith the transgenes when the genome insertions are performed. In eithersituation, the inserted transgene encoding the desired aPKMζ providesmore control of the modulation, particularly induction, of theexpression of the truncated form. This increased control enhances theability to alleviate memory defects and psychiatric dysfunctions.

[0039] A further manner of changing the amount of aPKMζ protein in anindividual is by administration of the protein itself to the individual.The protein is administered so that it is active in the central nervoussystem of the individual and thereby alleviates the memory defect orpsychiatric dysfunction by altering the memory formation component ofthe dysfunction. This protein can be an active or inhibitory form of themolecule.

[0040] This invention also relates to methods of identifying substancesthat affect memory formation or psychiatric dysfunctions in mammals,particularly in humans. The substance usually has an organic chemicalstructure and is in the form of a pharmaceutical with the requireddiluents, excipients and carriers present in its formulation. Thesubstance may be a macromolecule but usually it is much smaller. Inthese methods, the substance under consideration is administered to amammal. The administration is by any standard route. For example,administration can occur by oral or rectal intake, inhalation, topicalapplication, or parenterally by subcutaneous, intravenous orintramuscular injection. Once administered, it is determined whether thesubstance alters the expression or activity of an aPKMζ protein in thecentral nervous system of the mammal, where it has previously been shownthat the aPKMζ protein is associated with the psychiatric dysfunction ofinterest. The association of the aPKMζ protein and the psychiatricdysfunction can be direct or indirect. The association is present if analteration in the amount or activity of the aPKMζ protein eitherenhances or diminishes the signs or symptoms of the psychiatricdysfunction. The association is present if an alteration in the geneticexpression of the aPKMζ protein either enhances or diminishes the signsor symptoms of the psychiatric dysfunction. Alteration of expression oractivity of the aPKMζ protein is determined by comparing the expressionor activity of this protein after the substance is administered to themammal with the expression of the protein when the substance has notbeen administered. If a reproducible difference is found between theexpression or activity values for the aPKMζ protein when the substanceis present versus when the substance is not present, the substance isidentified as having the property of affecting the psychiatricdysfunction with which the aPKMζ protein is associated. The substancecan be further identified as having an alleviating or a deleteriouseffect on the psychiatric dysfunction, depending on the relationship ofthe expression change with the quality or intensity of the signs orsymptoms of the psychiatric dysfunction. For example, if an increase inthe expression or activity of the aPKMζ protein is associated with analleviation of the signs or symptoms of the psychiatric dysfunction andthe substance, when administered, causes an increase in the expressionor activity of the aPKMζ protein, the substance is considered to haveadvantageous properties for alleviating the psychiatric dysfunction.

[0041] Often for aPKMζ, when the administered substance causes anincrease in the expression or activity of the APKMζ, the substance isconsidered to have the property of enhancing the memory formationcomponent of the psychiatric dysfunction. Often, it is the short termmemory component of the psychiatric dysfunction that is enhanced whenthe administered substance causes an increase in the expression ofaPKMζ. Conversely, when the administered substance causes a decrease inthe expression or activity of the aPKMζ, the substance is considered tohave the property of interfering with the memory formation component ofthe psychiatric dysfunction. In this instance, it is again often theshort term memory formation component of the psychiatric dysfunctionthat is interfered with or blocked by the administered substance.

[0042] This invention further relates to methods for assessing theeffects of drugs on the memory formation component of psychiatricdysfunction. In these methods, the candidate drug is administered to anormal animal or an animal that possesses an inducible aPKMζ proteinwhich is associated with memory formation. The drug usually has anorganic chemical structure and is administered by any of the standardadministration routes together with any required diluents, excipients orcarriers. In these methods, the animal type is not limited to mammalsbut includes most of the animal kingdom. For example, insects such asDrosophila melanogaster or honeybees can be used as subjects forassessing the effects of drugs on the memory formation component. Othermodel organisms for assessing drug effects on memory formation includeC. elegans, Aplysia, Xenopus, zebrafish, mouse, rats, ferrets and cats.The only requirement for animal type is that it have an inducibletruncated form of an aPKCζ protein which is associated with memoryformation.

[0043] Following the administration of the candidate drug to the animalin these methods, the inducible aPKMζ protein is induced to produce thetruncated form in the animal, or the endogenous gene is examined in thenontransgenic animal. Induction can be performed by any of the methodsknown to persons who are familiar with such processes. In C. elegans,practitioners usually use heat-shock, antibiotics or small moleculessuch as IPTG. In Drosophila, practitioners usually use heat-shock,antibiotics, or small molecules like IPTG or heavy metals. In mammals,practitioners usually use antibiotics, hormones or small molecules likeIPTG.

[0044] After the expression of the aPKMζ protein has been induced in thetransgenic animal, or the endogenous gene in the normal animal, theanimal is subjected to a learning and memory assay, and a performanceindex, based on the outcome of the protocol, is assigned. Learning andmemory tests are known to psychologists and others who study learningand memory in animals. The training protocols that are useful in thisinvention address learning and memory attributes which the animalpossesses. Any discriminative classical conditioning protocol can beused. Exemplary of these protocols are associative and non-associativeconditioning protocols, classical and operant conditioning, and tests ofimplicit and explicit memory. The performance index is an assessment ofthe results of the training protocol that was used. Often theperformance index is a numerical value that is assigned by the observeror investigator to the outcome of the training protocol. The numericalvalue can be considered to be a scaled score for the performance of theanimal undergoing the classical conditioning protocol.

[0045] In these methods, the drug is considered to have an effect onmemory formation or the memory formation component of the psychiatricdysfunction when animals treated with the compound reproducibly performdifferently from untreated animals. In some instances, the sameindividual animal may serve as the control animal and the one to whomthe drug is administered. Usually, however, different animals serve asthe untreated (control) and treated (subject) animals. In theseinstances, the animals should be chosen from the same cohort. The drugsthat exhibit an effect on the memory formation component, as detected bythese methods, are candidates for administration to animals to alleviatepsychiatric dysfunctions in these animals, particularly when thepsychiatric dysfunction has a memory formation component.

[0046] In these methods, the inducible target of the drug is aPKMζ. Inthese instances, it is typically the short term memory formationcomponent of the memory problem or psychiatric dysfunction that isaffected by the drug. Memory problems targeted by the selected drugsinclude those resulting from normal aging, injury to the brain,Alzheimer's disease and neurodegeneration. Psychiatric dysfunctions forwhich the drugs selected by these methods will have an effect includeattention deficit disorder, autism, fragile X syndrome, bipolardisorder, schizophrenia, obsessive compulsive disorders, and phobias.

[0047] The study of PKC in memory formation has a long history. However,most previous studies were done before the complexity of the PKC genefamily was appreciated. The PKC family can be divided into three classesbased on their cofactor requirements. Whereas all PKC proteins requirephosphatidylserine for activation, the ‘conventional’ (cPKC) isotypesrequire diacylglycerol (DAG) and Ca2+ for full activity; ‘novel’ (nPKC)isotypes are Ca2+ independent but still require DAG, and the ‘atypical’(aPKC) isotypes are both DAG and Ca2+ independent. Structurally, thesekinases can be divided into an N-terminal regulatory domain, whichcontains a pseudosubstrate region as well as the binding sites for therequired cofactors, and the C-terminal catalytic domain. Removal of theN-terminal regulatory domain produces a persistently active kinase,referred to as PKM.

[0048] The roles of PKC in hippocampal models of synaptic plasticity,long-term potentiation (LTP), and long-term depression (LTD) have beenstudied extensively (see, F. Angenstein, et al., Prog.Neuropsychopharmacol. Biol. Psychiatry 21, 427-454 (1997)). Western blotanalyses with antibodies specific for each of the rat PKC isoformsdemonstrate that the only one whose levels specifically increase andremain elevated during the maintenance phase of LTP is PKMζ, thetruncated form of the a typical isozyme PKCζ(see, e.g., Osten, P. etal., J Neurosci. 16, 2404-2451 (1996)). Expression analyses also showthat the maintenance of LTD is associated with decreasing levels ofPKMζ. Most interestingly, LTP maintenance is abolished by sustainedapplication of low concentrations of the PKC inhibitor chelerythrine,whereas perfusion of PKMζ into CA1 pyramidal cells produces an increasein AMPA receptor-mediated synaptic transmission (D. S. F. Ling et al,unpublished data).

[0049] In Drosophila, the best characterized assay for associativelearning and memory is an odor-avoidance behavioral task (T. Tully, etal. J. Comp. Physiol. A157, 263-277 (1985) incorporated herein byreference). This classical (Pavlovian) conditioning involves exposingthe flies to two odors (the conditioned stimuli, or CS), one at a time,in succession. During one of these odor exposures (the CS+), the fliesare simultaneously subjected to electric shock (the unconditionedstimulus, or US), whereas exposure to the other odor (the CS−) lacksthis negative reinforcement. Following training, the flies are thenplaced at a ‘choice point’, where the odors come from oppositedirections, and expected to decide which odor to avoid. By convention,learning is defined as the fly's performance when testing occursimmediately after training. A single training trial produces stronglearning: a typical response is that >90% of the flies avoid the CS+.Performance of wild-type flies from this single-cycle training decaysover a roughly 24-hour period until flies once again distribute evenlybetween the two odors. Flies can also form long-lasting associativeolfactory memories, but normally this requires repetitive trainingregimens.

[0050] This task in Drosophila was used to examine in an exemplaryfashion herein the role of a typical PKM in memory formation. Inductionof the mouse aPKMζ (MaPKMζ) transgene enhances memory, and corrects thememory defect of radish mutants. There is a single a typical PKC inDrosophila (http://www.fruitfly.org), and the truncated ‘M’ isoform,DaPKM, was found to be preferentially expressed and active in fly heads.Both pharmacological and dominant-negative genetic intervention ofDaPKC/M activity disrupt normal memory. Finally, induction of thepredicted DaPKM also enhances memory, further demonstrating a generalrole of aPKM in memory processes.

[0051] A description of preferred embodiments of the invention follows.

EXEMPLIFICATIONS

[0052] Methods

[0053] Fly Stocks and Maintenance.

[0054] The background stock (2202 u) used as wild-type flies in all theexperiments is w (isoCJ1), which is an isogenic line derived from aw¹¹¹⁸line backcrossed repeatedly to a Canton-S wildtype strain. Tominimize differences in genetic background, 2202 u also served as therecipient strain for all of the transgenic lines used in theseexperiments. Fly stocks used for behavioral analyses were maintainedunder appropriate conditions.

[0055] Pavlovian learning and memory in Drosophila. To assess learningand memory in Drosophila, an olfactory-avoidance classical (Pavlovian)conditioning protocol was used. This protocol was modified to facilitateautomated and repetitive training regimens. 3-octanol (OCT) and4-methylcyclohexanol (MCH) were used as odors in these experiments.Detailed descriptions of single-cycle massed and spaced training as wellas testing and the tests for olfactory acuity and shock reactivity canbe found in Tully et al., Cell 79, 35-47 (1994) or Connolly, J. B. &Tully T., Drosophila: A Practical Approach (ed. Roberts, D. B., OxfordUniv. Press, Oxford 1998) incorporated herein by reference. Theperformance index (PI) was calculated by subtracting the number of fliesmaking the incorrect choice from those making the correct one, dividingby the total number of flies, and multiplying by 100. To avoidodor-avoidance biases, the PI of each single n was calculated by takingan average performance of two groups of flies, one group trained withthe CS+ being OCT, the other with the CS+ being MCH.

[0056] Transgenes

[0057] The MaPKMζ DNA construct was produced using PCR amplificationusing the full-length, MaPKCζ cDNA (American Type Culture Collection(ATCC) no.63247) as the template. PCR primers (upstream primer:5′-CTAGCGAATTCAACATGAAGCT GCTGGTCCATAAACG-3′ (SEQ ID NO. 1); downstreamprimer: 5′-CTAGCTCTAG ATCACACGG ACTCCTCAGC-3′ SEQ ID NO. 2) were used toproduce the truncated MaPKMζ gene. The upstream primer contained anEcoRI restriction site just 5′ of a consensus Kozak sequence and alsoencoded an ATG start codon. The last 20 nucleotides of the upstreamprimer correspond to 20 nucleotides in the hinge region of the MaPKCζgene. The second codon in this truncated gene corresponds to amino acid165 in the MaPKCζ protein. The downstream primer is antisense to thelast 20 nucleotides in the MaPKCζ open reading frame, and contains aXbaI restriction enzyme site immediately after the translation stopcodon. The PCR product produced using these primers was cut with EcoRIand XbaI, then subcloned into a heat-shock P-element vector, using thesame restriction sites, and sequenced. The kinase-inactive KI-MaPKMζ wasproduced in the same manner except that the K281W-MaPKCζ mutant DNA wasused as the template for PCR amplification. The full-length MaPKCζ genewas subcloned into the same P-element vector using the EcoRI siteslocated at both ends of the cDNA and in the heat-shock vector.Transgenic flies were made using standard techniques. Based on homologywith mammalian PKCζs, the N terminus of DaPKC was defined as beginningwith residues M-P-S. Using this reference point, the DaPKM transgenebegins at Met223 within the hinge region of DaPKC.

[0058] Heat-Shock Induction

[0059] All fly stocks were maintained at 25° C. before and afterheat-shock. Heat-shock inductions were performed in 15-ml plastic tubes(˜100 flies/tube) by partially submerging the tubes in a waterbath atthe appropriate temperature (32° C. or 37° C.) for 30 min.

[0060] Biochemical Assay

[0061] MaPKMζ transgenic flies maintained at 25° C. were heat-shocked at37° C. for 30 min, then allowed to recover at 25° C. for 1 h and frozenin liquid nitrogen. Uninduced controls and wild-type flies remained at25° C. throughout. After freezing, fly heads were separated from bodiesand ˜100 μl of heads were homogenized in 1 ml extraction buffer (20 mMHepes pH 7.4, 0.2 M sucrose, 1 mM EDTA, 1 mM EGTA, 1 mMphenylmethylsulfonyl fluoride (PMSF), and Complete protease inhibitorcocktail (Roche, Indianapolis, Ind.)), and centrifuged at 176,500 g for10 min. The homogenates were diluted to 1 mg/ml protein and adjusted to2.5 mM EGTA. PKC activity was assayed by measuring the incorporation of³²P from γ-³²P-ATP into peptide-ε_(Peninsula, Belmont, Calif.). Each50-μl reaction contained 50 mM Hepes pH 7.4, 10 mM MgCl₂, 1 nMdithiothreitol (DTT), 25 μM peptide-ε, 20 μg of protein homogenate, and20 mM γ-³²P-ATP(2 μCi). The protein was incubated at 26° C. for 1 min inthe reaction mixture lacking ATP. The reaction was started by theaddition of γ-³²P-ATP, incubated at 26° C. for 2 min and stopped on icewith 20 μl of 75 mM H₃PO₄. A 20-μl aliquot from each reaction wasspotted onto a 2-cm circle of P81 chromatography paper (GIBCO,Gaithersburg, Md.) and washed three times in 75 mM H₃PO₄ dried, and theradioactivity measured by scintillation counting. The effect of Ca2+ andphorbol esters in the reaction was measured in the presence of 200 μMCaCl₂ and 400 μM phorbol myristate acetate (PMA). Background activitywas estimated from mock reactions lacking peptide-ε. PKC activityincreased linearly with time up to 4 min. The values reported areaverages from quadruplicate assays.

[0062] Western Blots

[0063] The heat-shock induction time course was monitored using fly-headextracts (approximately five heads per lane were loaded) for the westernanalyses. A rabbit polyclonal antibody against MaPKMζ (Sigma, St. Louis,Mo.) was used as the primary antibody, and the protein was detectedusing ECL (Pierce, Rockford, Ill.).

[0064] Chelerythrine Feeding

[0065] A 4% (wt/vol) sucrose solution was used as a vehicle for thechelerythrine feeding. Onto Whatman 3MM filter paper cut to fit andplaced in the bottom of standard fly culture vials, 120 μl of 0 μM(sucrose alone), or 25 μM, 50 μM or 100 μM chelerythrine was applied.Flies were starved in empty vials (˜100 flies/vial) for 1 h, and thentransferred to the feeding vials for 3 h to allow sufficient feeding.Flies were then trained and tested as described in the text and figures.Feeding was monitored by placing 1/50 volume of green food coloring intothe solutions, which could then be seen in the abdomens of flies aftereating. There was no noticeable difference in consumption between any ofthe solutions.

[0066] Results

[0067] MaPKMζ Induction Enhances Memory in Drosophila

[0068] To investigate the role of PKC in learning and memory inDrosophila, transgenic lines of flies were made bearing heatshock-inducible murine a typical PKC (MaPKC) isoforms. Considering thatLTP experiments indicate that MaPKMζ levels increase after thepresentation of the stimuli required for long-lasting potentiation,inducing MaPKMζ after training was tested to see if it affectedolfactory memory. Induction by mild heat shock (32° C.) after trainingstrongly enhanced 24-hour memory (FIG. 1a). This enhancement was not dueto transgene-independent heat-shock effects, because the wild-type fliesdid not show enhanced memory when exposed to heat shock. The transgenicflies were made in this wild-type strain, so the enhancement was not dueto differences in genetic background. Finally, the memory enhancementdid not result from an insertional mutation caused by the transgene,because two independent lines (MaPKMζ-14 and MaPKMζ-43) had similareffects.

[0069] Enhancement of 24-hour memory after single-cycle training byinducing MaPKMζ with a strong heat shock (37° C.) 3 hours beforetraining was also tested, but this regimen had no effect (data notshown). Because transgene induction after behavioral training enhancedmemory, whereas induction before training did not, the temporalspecificity of this MaPKMζ dependent effect was next examined. Optimalenhancement was found to occur when heat-shock induction begins 30minutes after training ends, and the effect is absent if heat shockoccurs before, or is delayed until 2 hours after training (FIG. 1b).

[0070] The memory enhancement was not observed when a kinase-inactive(KI) mutant of MaPKMζ was induced either before or after training (FIG.2a; two independent lines, KI-1 and KI-2, of KI-MaPKMζ). The enhancementwas also not observed when full-length (FL)-MaPKCζ was induced before orafter training (FIG. 2a; two independent lines, FL-3A and FL-14A). Thefailure of either the KJ-MaPKMζ or the FL-MaP-KCζ transgene to enhancememory was not due to lack of expression, because both are expressed atlevels comparable to the MaPKMζ protein (FIG. 3a; KI-1 and FL-14A versusMaPKMζ-14). Together, these results indicate that the memory enhancement(FIG. 1) requires a persistently active aPKM isoform.

[0071] Biochemical Detection of MaPKMζ Induction in Drosophila

[0072] Inducible increases in MaPKMζ protein levels and kinase activitywere detected in extracts made from Drosophila heads (FIG. 3). Westernblot analyses showed that both the mild and strong heat-shock regimensinduced the MaPKMζ and MaPKCζ isoforms, and that these proteinspersisted for ˜18 hours after heat shock (FIG. 3a). The induced MaPKMζprotein was active, as indicated by an observed enhancement ofCa²⁺/DAG(diacylglycerol)-independent PKC activity in fly head extractsfrom induced but not from uninduced transgenic flies (FIG. 3b).

[0073] MaPKMζ Induction Does Not Affect Peripheral Behaviors

[0074] The memory enhancement occurred only when the transgene wasinduced after training; therefore it is not likely due to an effect oftransgene expression on the perception of either the shock or the odorsat the time of training. No effect of MaPKMζ induction on shockreactivity was found, as the transgenic flies behaved indistinguishablyfrom the wild-type strain, irrespective of heat shock (FIG. 2b). Thus,transgenic flies did not perceive shock better during training. Becausethe memory enhancement was induction-dependent (FIG. 1a), it cannot beattributed to small amounts of leaky expression during training.Although MAPKMζ had decayed to pre-heat-shock levels by the time oftesting at 24 hours (FIG. 3a), enhancement of olfactory responses at thetime of testing by MaPKMζ could be occurring. However, this was not thecase because olfactory acuity at 24 hours after induction was normal(FIG. 2b ). These data demonstrate that the MaPKMζ induction has nobehavioral effect on either sensory modality, and indicate that theeffect observed is due to bona fide memory enhancement.

[0075] MaPKMζ Enhances Memory After Massed, but not Spaced Training

[0076] Drosophila can form associative olfactory memories lasting 24hours and longer, but this normally requires repetitive training.Multiple-trial training regimens have been established that produce bothanesthesia-resistant memory (ARM) and long-term memory (LTM). ARM can beproduced by 10 cycles of ‘massed’ training with no rest intervalsbetween the individual training trials, and lasts 2-3 days. LTM resultsfrom repetitive training that contain rest intervals (15 min each; seeMethods), and 10 cycles of this ‘spaced’ training generates LTM thatlasts at least 7 days. To test whether MaPKMζ could enhance ARM or LTM,flies were subjected to massed or spaced training regimens, thetransgene was induced for 30 minutes after training, and then 4-daymemory was measured.

[0077] MaPKMζ induction substantially increased 4-day memory aftermassed training (FIG. 4a; HS+ compared to −HS for each line) but did notimprove 4-day memory after spaced training (FIG. 4b; HS+ compared to −HSfor each line). These data indicate that MaPKMζ induction enhancesmassed training-induced, but not spaced training-induced memory.

[0078] The Radish Mutation Does Not Block MaPKMζ-Induced MemoryEnhancement

[0079] Previous work indicated that consolidated memory in Drosophilaconsists of two biochemically separable components: ARM and LTM. ARM isproduced by either massed or spaced training and it is insensitive tocycloheximide treatment. LTM is produced by spaced training and isblocked by cycloheximide treatment; thus it is considered to requireacute protein synthesis. A previously identified Drosophila memorymutant, radish, is deficient in ARM, as this mutation blocks memoryproduced by massed training. Spaced training of radish mutants doesproduce memory, but this memory can be completely blocked by treatingthe mutants with cydoheximide. These results led to a two-pathway modelof consolidated memory, one dependent on the radish gene product (ARM)and the other dependent on activity-induced, acute protein synthesis(LTM). (See Tully et al, Cell 79, 35-47 (1994).

[0080] Because MaPKMζ induction enhanced memory after massed but notafter spaced training, the dependence of this effect on radish wastested. The radish gene is on the X chromosome in Drosophila andhomozygous radish mutant females were crossed to males homozygous for anautosomal copy of the heat shock-inducible MaPKMζ transgene. The radishmutant is recessive, thus the heterozygous female progeny of this matingwill have normal memory after massed training, whereas the hemizygousmales will display the radish memory deficit in the absence ofinduction. The progeny were subjected to massed training, followed bythe standard MAPKMζ induction after training, and then tested at 24hours to assess the ability of MaPKMζ. Males and females were trainedand tested en masse and then separated and counted. The radish mutationdid not block the memory effect of MaPKMζ induction (FIG. 5). The memorydefect of radish males was apparent in the absence of heat-shockinduction (HS−), but memory was clearly present in induced males (HS+).A lesser, but significant induction-dependent memory enhancement of theheterozygous radish females by MAPKMζ was also observed (FIG. 5:HS+versus HS− females).

[0081] A Drosophila Homolog of MaPKMζ

[0082] There is a single a typical PKC (DaPKC) gene in the Drosophilagenome (http://www.fruitfly.org), and it is highly homologous to theMaPKCζ gene used. (The kinase domain shows 76% identity and 87%similarity.) A western blot of extracts made from wild-type fly headsand bodies showed that the antiserum used to detect MaPKMζ and MaPKCζrecognized two bands in fly extracts, the smaller of which was enrichedin head extracts (FIG. 6a). This antiserum is directed against theC-terminal 16 amino acids of MaPKC/Mζ, which shares substantial homologywith DaPKC. Antiserum from mice immunized with peptides derived fromDaPKC recognized these same bands (data not shown). The molecularweights of these two bands indicate that they are probably the DaPKC(˜73 kDa) and DaPKM (˜55 kDa) isoforms.

[0083] We have not established the N-terminal sequence of the lowermolecular weight band; however, it likely represents an endogenous DaPKMisoform. The immunoreactivity was competitively reduced by a peptidefrom the corresponding region of DaPKC, but not one outside of thisepitope (FIG. 6b, 590 and 545, respectively) or by a peptide fromanother Drosophila protein (dCREB2, data not shown). In agreement withthe western blot data, fly heads contained more Ca2+ and DAG-independentPKC activity than did bodies (FIG. 6c). The presence of the putativeDaPKM correlates strongly with this enriched activity, suggesting thatmost, if not all, of the endogenous a typical kinase activity wemeasured in head extracts was due to this DaPKM isoform. These dataindicate that flies possess both ‘C’ and ‘M’ forms of an a typical PKCthat is highly homologous to MaPKC/M, and that the DaPKM is enriched inheads.

[0084] Chelerythrine and KI-MaPKMζ Inhibit Memory, but not Learning

[0085] A P-element insertional mutant in DaPKC has been described;however, it is an embryonic lethal and thus is not suitable forexamining a possible role in adult learning and memory formation. Toassess whether this gene's product is necessary for memory formation,two approaches were taken. First, the effects on memory of feeding fliesthe PKC inhibitor chelerythrine were monitored. This drug is reported toselectively inhibit PKMζ at low concentrations (D. S. F. Ling et al.,unpublished data and Laudanna, C. et al, J. Biol. Chem. 273, 30306-30315(1998); however, its specificity is controversial, and it inhibits otherPKC isotypes at higher concentrations. Measurements were also made ofmemory effects produced by inducing the kinase-inactive KI-MaPKMζprotein, which displays ‘dominant-negative’ activity that is likely tobe specific to the a typical PKCs, leaving cPKC and nPKC responsesintact.

[0086] Feeding flies chelerythrine inhibited 24-hour memory formation ina dose-dependent manner (FIG. 7a), and induction of the KI-MaPKMζinhibited 24-hour memory after massed training (FIG. 7b). The inhibitoryeffects of both chelerythrine and the KI-MaPKMζ were not likely due toeffects on olfactory acuity or shock reactivity because learning wasunaffected by either treatment (FIG. 7c).

[0087] DaPKM Induction Enhances Memory

[0088] The memory enhancement produced by MaPKMζ could have been due toproperties unique to this mammalian protein. The expression data showingthat DaPKM was expressed and active in Drosophila heads, when combinedwith the chelerythrine and dominant-negative data, suggested that DaPKMis involved in normal memory processes in Drosophila. The extensivestructural homology between MaPKMζ and DaPKM also argued againstfunctional uniqueness. An hypothesis of functional homology makes astrong prediction: induction of DaPKM after training should also enhancememory.

[0089] Using as a basis the approximate molecular weight of the DaPKM,the DAPKC gene was truncated within the hinge region separating theregulatory from the catalytic domains such that the putative DaPKM genebegins at methionine 223. Induction of the DaPKM transgene aftertraining enhanced 24 hour memory after single-cycle training (FIG. 8a).One of these lines was used to show that 4-day memory after massedtraining was also enhanced (FIG. 8b). As with the MaPKMζ transgenes, theDaPKM lines showed rapid heat-shock induction (FIG. 8c). These resultsconfirm those obtained with MaPKMζ, and thus indicate that aPKM isfundamental in the mechanisms underlying memory across species.

[0090] Discussion of Results

[0091] Atypical PKM and Normal Memory

[0092] These results provide strong evidence that a typical PKM activityis sufficient to enhance memory in Drosophila. Both pharmacological anddominant-negative interventions were investigated. Chelerythrineinhibited normal memory in a dose-dependent manner (FIG. 7a), andinduction of a predicted dominant-negative a typical PKM produced thesame memory deficit (FIG. 7b ).

[0093] It is important to note that neither of the employed inhibitoryinterventions disrupted learning (FIG. 7c). Screens in Drosophila haveidentified many learning mutants that disrupt several signaling pathways(e.g., cAMP-PKA, integrin-mediated, and 14-3-3 protein-dependentprocesses; for original citations). Considering that learning remainsnormal, it is unlikely that either intervention produced very broadsignaling defects. With this in mind, the observation that eachintervention inhibited memory without disrupting learning indicates thatDaPKC/M is a component of an endogenous memory mechanism.

[0094] Phase Specificity of MaPKMζ-Enhanced Memory

[0095] Heat-shock induction of MaPKMζ did not enhance long-term memory,because it did not improve memory after spaced training (FIG. 4b). Oneexplanation for this is that spaced training induces endogenousmaintenance mechanisms, and thus occludes the effect of inducing theMaPKMζ transgene. Thus, memory after single-cycle or massed training maybe prolonged by transgene induction because these training regimens donot normally induce prolonged a typical PKM activity. Work in honeybeesshowed that single-cycle training produces neither persistent PKCactivity nor long-lasting memory, but multiple-cycle training producesboth (see L. Grunbaum et al, J. Neurosci. 18, 4384-4392 (1998)). Thememory enhancement observed when inducing MaPKMζ may simply bypass theendogenous requirements (normally provided by spaced training) forprolonged activation of aPKM.

[0096] The MaPKMζ-induced enhancement of massed, but not spaced trainingprompted an examination the involvement of the radish gene product inthis process. If radish were required for the enhancement, the radishmutation would have blocked the MAPKMζ-induced effect, and this wasclearly not the case (FIG. 5). Although MaPKMζ induction phenotypicallyrescues the memory defect of radish, it does not do so because radishencodes for the Drosophila aPKM. DaPKM is on the second chromosome andradish is on the X, and no Drosophila PKC gene maps to the geneticallydefined radish locus. There are two principal possibilities explaininghow MaPKMζ-induced memory enhancement bypasses the defect of radishmutants: (1) MaPKMζ is downstream of radish; (2) MaPKMζ activates apathway that is parallel to and independent of radish. The firstinterpretation is favored because either enhancement or disruption ofmemory after massed training, as well as partial rescue of the radishphenotype can be carried out.

[0097] Atypical PKM and activity-dependent synaptic plasticity. Thereare two general interpretations of these data: PKMζ acts to increaseeither (1) the magnitude or (2) the duration of the synapticpotentiation that underlies the behavior. In the first model, PKMζenhances the synaptic machinery induced by training, making a ‘stronger’synaptic connection that decays more slowly. In the second model, PKMζacts solely to maintain the synapses previously modified by experience,with no effect on the induction of the potentiation. If one considersthe behavioral measurements of learning (testing done immediately aftertraining) and memory (testing done after a longer time) with inductionand maintenance, respectively, the chelerythrine and dominant-negativedata argue for a role in maintenance. Neither of these treatmentsaffected learning (FIG. 7c), but each inhibited memory (FIG. 7a and b).An enhancement of learning by prior induction of PKMζ was not detected(data not shown), nor was there an improvement of 3-hour memory if PKMζwas induced 30 minutes after training (data not shown). Although themagnitude and duration models may be artificially exclusive, takentogether the data are most consistent with a role of PKMζ in themaintenance of experience-dependent synaptic plasticity.

[0098] The stability of a synapse varies in response to differentregimens of stimuli. Long-lasting changes normally require multiplestimuli and depend on new protein synthesis. Recent experiments supportthe existence of a synaptic marking system that enables neurons to tagrecently active synapses, thus maintaining synaptic specificity duringthe cell-wide process of protein synthesis-dependent long-term memoryformation (see, e.g., U. Frey et al., Nature 385, 533-536 (1997)). Asynapse that would normally be stable for only a short period of timecan be potentiated for a much longer period of time. However, to do soit must be activated within 2-4 hours of stimulation that produceslong-term changes at a second and separate synapse within the sameneuron. Although no direct evidence for a role of PKMζ in this processwas shown by these results, the similarity between the temporal windowsfor the proposed synaptic tag and the memory enhancement observed heresuggest a mechanistic relationship between them.

[0099] DaPKC is part of a multiprotein complex important for both cellpolarity and the asymmetrical cell divisions of early Drosophilaneurogenesis (see, e.g., A. Wodarz et al, J. Cell Biol. 50, 1361-1374(2000)). These processes show strong structural and functional parallelswith the first asymmetrical cell division of Caenorhabditis elegansembryogenesis. The Drosophila homologs of C. elegans proteins importantfor this process, Par-3 (Bazooka) and Par-6 (DmPar-6), interact witheach other and with DaPKC to direct a specific and interdependentsubcellular localization of the complex. During early Drosophilaembryogenesis, Bazooka, DmPar-6, and DaPKC are localized to the zonulaadherens, a cell junction structure. Mutation in any one of these genesdisrupts the ability of the remaining two proteins to localize to thisstructure properly, and this disrupts cell polarity. This mutualdependence for localization is also apparent during neurogenesis, andcauses the inappropriate segregation of cell determinants. Thismultiprotein complex is critical in mammalian cell polarity and inorganizing junctions between epithelial cells. The mouse homologs ofBazooka and Par-6 are expressed in various regions of the CNS, and theirsubcellular localization within CA1 hippocampal neurons is consistentwith a role in synaptic plasticity (see D. Lin et al, Nature Cell Biol.2, 540-547 (2000)). Bazooka and DmPar-6 are expressed in Drosophilaheads, as are DaPKC and DaPKM (FIG. 6a).

[0100] Atypical PKM has been shown here to be sufficient to enhancememory in Drosophila, and the chelerythrine and dominant-negative datasuggest that it is also necessary for normal memory.

[0101] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the scope of the inventionencompassed by the appended claims.

1 2 1 37 DNA Artificial Sequence PCR Primer 1 ctagcgaatt caacatgaagctgctggtcc ataaacg 37 2 29 DNA Artificial Sequence PCR Primer 2ctagctctag atcacacgga ctcctcagc 29

What is claimed is:
 1. A method of alleviating a memory problem or Apsychiatric dysfunction in a mammal comprising modulation the expressionof a truncated form of an aPKCζ protein in the central nervous system ofsaid mammal, wherein said modulation results in an alleviation of saidpsychiatric dysfunction in said mammal.
 2. The method of claim 1 whereinsaid truncated form of an aPKCζ is aPKMζ.
 3. The method of claim 2wherein said modulation is either the induction of formation of an aPKMζprotein in the central nervous system of said mammal or the inhibitionof formation of an aPKMζ protein in the central nervous system of saidmammal.
 4. The method of claim 3 wherein the memory problem is selectedfrom the group consisting of normal aging, injury to the brain,Alzheimer's disease, or neurodegeneration, and said psychiatricdysfunction is a disorder selected from the group consisting ofattention deficit disorder, autism, fragile X syndrome, bipolardisorder, schizophrenia, obsessive compulsive disorder, and phobias. 5.The method of claim 4 wherein said induction enhances the memoryformation component of said memory problem or said psychiatricdysfunction and said inhibition interferes with the memory formationcomponent of said memory problem or said psychiatric dysfunction.
 6. Themethod of claim 5 wherein said memory formation component is short termmemory formation.
 7. The method of claim 6 wherein said mammal is ahuman.
 8. The method of claim 7 wherein said modulation is the inductionof an aPKMζ which enhances memory formation.
 9. The method of claim 5wherein said induction occurs on a transgene that has been introducedinto the individual for whom enhanced memory is sought.
 10. A method ofidentifying a substance that affects a memory problem or a psychiatricdysfunction in a mammal comprising: (a) administering said substance tosaid mammal; (b) determining whether said substance alters theexpression or activity of a truncated form of an aPKCζ protein in thecentral nervous system of said mammal when compared to the expression oractivity of said truncated form of said aPKCζ protein in the centralnervous system in the absence of said substance, wherein said truncatedform of said aPKCζ protein is associated with said memory problem orsaid psychiatric dysfunction in said mammal; and (c) indicating thatsaid substance affects said memory problem or said psychiatricdysfunction when the expression or activity of said truncated form ofsaid aPKCζ protein in the presence of said substance differs from theexpression or activity of said truncated form of said aPKCζ protein inthe absence of said substance.
 11. The method of claim 10 wherein saidtruncated form of said aPKCζ is aPKMζ.
 12. The method of claim 11wherein the memory problem results from normal aging, injury to thebrain, alzheimer's disease or neurodegeneration, and the psychiatricdysfunction is a disorder selected from the group consisting ofattention deficit disorder, autism, fragile X syndrome, bipolardisorder, schizophrenia, obsessive compulsive disorder and phobias. 13.The method of claim 12 wherein an increase in the expression or activityof said aPKMζ when said substance is present, compared to the expressionor activity of said aPKMζ when said substance is not present, isindicative of an enhancement of memory formation of said memory problemor the memory formation component of said psychiatric dysfunction. 14.The method of claim 13 wherein said memory formation component is shortterm memory formation.
 15. The method of claim 14 wherein a decrease inthe expression or activity of said aPKMζ when said substance is present,compared to the expression or activity of said aPKMζ when said substanceis not present, is indicative of an interference in normal memoryformation or the memory formation component of said psychiatricdysfunction.
 16. A method of increasing memory formation or the memoryformation component of a psychiatric dysfunction in a mammal comprisingincreasing the functional level of an aPKMζ from normal in said mammal,wherein said aPKMζ is associated with memory formation or said memoryformation component in said mammal, and wherein said increase in memoryformation is sought to alleviate a memory problem selected from thegroup consisting of normal aging, injury to the brain, Alzheimer'sdisease, or neurodegeneration, and said psychiatric dysfunction is adisorder selected from the group consisting of attention deficitdisorder, autism, fragile X syndrome, bipolar disorder, schizophrenia,obsessive compulsive disorder and phobias.
 17. A method of increasingmemory in an animal comprising increasing the functional level oractivity of aPKMζ from normal in said animal, wherein said aPKMζ isassociated with memory formation in said animal.
 18. A method forassessing the effect of a drug on normal memory formation or the memoryformation component of a psychiatric dysfunction comprising: (a)administering said drug to an animal having an inducible truncated formof an aPKCζ protein associated with memory formation; (b) inducingexpression of said truncated form of said aPKCζ protein; (c) subjectingsaid animal to a classical conditioning protocol; and (d) assessing theperformance index of said classical conditioning, wherein said drug hasan effect when said drug alters said performance index from theperformance index value obtained by said animal in the absence of saiddrug.
 19. The method of claim 18 wherein said truncated form of saidaPKCζ is aPKMζ.
 20. The method of claim 19 wherein memory formation isassociated with a memory problem selected from the group consisting ofnormal aging, injury to the brain, alzheimer's disease orneurodegeneration, and said psychiatric dysfunction is a disorderselected from the group consisting of attention deficit disorder,Alzheimer's disease, autism, fragile X syndrome, bipolar disorder,schizophrenia, obsessive compulsive disorder, and phobias.
 21. Themethod of claim 20 wherein said memory formation or said memoryformation component is short term memory formation.
 22. The method ofclaim 19 wherein said animal is Drosophila.
 23. The method of claim 19wherein said drug acts on a transgene that has been introduced into saidanimal.
 24. A method of alleviating a memory problem or psychiatricdysfunction in a mammal comprising modulating the expression or activityin said mammal of a gene that encodes a truncated form of an aPKCζprotein associated with said psychiatric dysfunction.
 25. The method ofclaim 24 wherein said gene that encodes said truncated form of saidaPKCζ protein is an aPKMζ gene.
 26. The method of claim 25 wherein saidmemory problem is selected from the group consisting of normal aging,injury to the brain, Alzheimer's disease, or neurodegeneration, and saidpsychiatric dysfunction is a disorder selected from the group consistingof attention deficit disorder, autism, fragile X syndrome, bipolardisorder, schizophrenia, obsessive compulsive disorder, and phobias. 27.The method of claim 26 wherein said modulation is an induction of saidgene that encodes said aPKMζ protein and said induction enhances memoryformation associated with said memory problem or the memory formationcomponent of said psychiatric dysfunction.
 28. The method of claim 27wherein said memory formation or said memory formation component isshort term memory formation.